We plan to synthesize the 5-deaza analogues of riboflavin, FMN, and FAD, in which a carbon replaces N5 of the isoalloxazine ring of the coenzymes, as specific probes of flavoenzymes. We believe the deazaflavin molecules provide singular opportunities for novel insights into how flavoenzymes oxidize substrates, interact with specific electron acceptors and control the mechanistic and stereochemical pathways open to the coenzyme. 5-Deazariboflavin undergoes enzyme-catalyzed, stereospecific reduction by NADH in the presence of a marine bacterial oxidoreductase. With 4-R-(H3)-NADH, direct hydrogen transfer to 5-dRFH2 has been established. Similarly, yeast Old Yellow Enzyme and M. smegmatis L-lactate oxidase reduce 5-dFMN. The questions of hydride transfer and stereochemical categorization of flavoenzymes will now be evaluated. With L-hydroxy acid oxidases and D-amino acid oxidase, the deaza analogues should shed light on the role the flavin plays in recently discovered alpha, beta-eliminations carried out by these enzymes. Also the susceptibility of the dFMN- or dFAD-holoenzymes to the suicide substrates, hydroxybutynoate and nitroethane respectively, will be probed. With OYE and DAAO, we will determine whether the green charge transfer complexes which form with the normal coenzymes also form when the deaza analogues are used.